This project is an integrated effort of chemistry, structural biology, and bacterial genetics with a focus on the enzyme LuxS. LuxS is responsible for producing the key quorum sensing molecules, AI-2, in many different species of pathogenic bacteria. A long range goal is a more complete understanding of the inter- species quorum sensing function with the implication of a new approach to control the pathogenicity of some of the most serious bacterial pathogens, such as Vibrio cholerae and Bacillus anthracis. LuxS produces DPD, the source of spontaneously interconverting molecules and borate diesters that are collectively called AI-2. Design, synthesis, and evaluation of substrate analog, transition state analog, and mechanism-based inhibitors of LuxS is a major goal of this project. The designs will be based on protein structural information and the proposed mechanism of action of LuxS. The designed inhibitors will be optimized for activity in vitro, and then refined for effectiveness and favorable pharmacokinetics in vivo. Effective inhibitors will be crystallized with native LuxS to provide a detailed structure of the active enzyme/inhibitor interaction. High throughput screening of compound libraries will be undertaken in collaboration with the Broad Institute, using a well-established assays for LuxS activity in vivo and in vitro. The goal is to identify new structural types that can function as inhibitors of LuxS. An X-ray crystallographic effort will provide information on the interaction of new inhibitors with LuxS, and this information will feed back into the inhibitor design. After initial evaluation in the highly sensitive bioluminescent Vibrio harveyi bioassay, promising inhibitors will be tested in the two clinically important pathogens, Salmonella typhimurium and Vibrio cholerae. These studies may result in the development of novel broad-spectrum bacterial control agents. Working by a new principle that does not kill the bacteria, this approach is unlikely to suffer from the rapid development of resistance as observed with most traditional anti-bacterials. This project aims to discover new small molecules that will inhibit quorum sensing in bacteria through the inhibition of the AI-2 signaling system. The AI-2 system appears to control pathogenicity over a broad spectrum of bacteria, and potent inhibitors will serve as probes to investigate this system in different species as well as in multi-species colonies. Potent inhibitors with appropriate pharmacological properties will be candidates for an entirely new approach to control of pathogenic bacteria, as a supplement or replacement for conventional antibiotic therapy. [unreadable] [unreadable] [unreadable]